Human Dendritic Cells Transmit Enterovirus A71 via Heparan Sulfates to Target Cells Independent of Viral Replication

ABSTRACT Enterovirus A71 (EV-A71) is a causative agent of life-threatening neurological diseases in young children. EV-A71 is highly infectious but it remains unclear how the virus disseminates from primary entry sites—the mucosa of the respiratory tract or the intestine—to secondary replication sites—skin or brain. Here, we investigated the role of dendritic cells (DCs) in EV-A71 dissemination. DCs reside in the mucosa of the airway and gut, and migrate to lymphoid tissues upon activation and, therefore, could facilitate EV-A71 dissemination to secondary replication sites. Monocyte-derived DCs were not permissive to different genotypes of EV-A71 but, notably, coculture with EV-A71-susceptiblle RD99 cells led to very efficient infection of RD99 cells. Notably, EV-A71 transmission of DCs to RD99 was independent of viral replication as a replication inhibitor did not affect transmission. Soluble heparin blocked EV-A71 transmission by DCs to RD99 cells, in contrast to antibodies against known attachment receptor DC-SIGN. These results strongly suggest that DCs might be a first target for EV-A71 and involved in viral dissemination via heparan sulfates and heparin derivatives might be an effective treatment to attenuate dissemination. IMPORTANCE EV-A71 is an emerging neurotropic virus that is of emerging concern and can result in polio-like illness. The exact mechanism of how EV-A71 results in neurological symptoms are unknown. In particular, the early dissemination of the virus from primary replication sites (airway and intestine) to secondary sites (central nervous system and skin) needs to be elucidated. There is evidence pointing toward a role for dendritic cells (DC) in EV-A71 transmission. Moreover, heparan sulfate (HS) binding mutations are observed in patients with severe diseases. Therefore, we evaluated the potential role of HS on DC in transmission. We find that HS are critical for transmitting EV-A71 by DC to target cells. Our data are consistent with other clinical and in vitro observations highlighting the importance of HS in EV-A71-induced disease.

marker CD1a might not serve as the marker for the EV-A71-infected DCs. To clarify the absence of EV-A71 infection in DCs, the authors should add the data of only DCs, thus in the absence of RD99 cells, in the context of experiments in Fig.1. 3. L136: CD1a?
Reviewer #2 (Comments for the Author): Helgers et al present a study investigating the role of dendritic cells (DC's) in the transmission of EV-A71. Using a combination of flow cytometry and qRT-PCR the authors demonstrate that, while not susceptible to EV-A71 infection themselves, DC's are capable of transmitting infectious virus to susceptible cells following co-culture, and doing so in a replication-independent manner. They subsequently demonstrate a role for heparan sulfates in this process by inhibiting transmission with soluble heparin. This is an interesting study that suggests a potential mechanism by which EV-A71 is able to establish secondary sites of infection. However, I have some questions/comments to be addressed: Major 1. Did the authors carry out any characterisation of the DC population following differentiation but prior to use in co-culture? DC populations can vary widely between donors and it would be good to include, at minimum, the details of how the DC populations used in co-culture were initially defined. Minor 1. Line 111: Could the authors clarify the RT reagent used as SupersScript II is not a Promega product. 2. Line 134: Remove extra full stop after RD99 cells. 3. Line 136: Could the authors clarify the target used to determine the DC cell population? In the Methods and Figure, cells are separated on the basis of CD1a expression, but the authors state here that DC populations were distinguished by DC-SIGN. 4. Line 185-187: The authors discuss assessing a role for PSGL-1 in DC-mediated transmission of EV-A71. However, there is no data or details in the results on PSGL-1, which instead focus on DC-SIGN and heparan sulfates. Do the authors have this data to include in the manuscript? If not reference to this should be removed from the discussion for the sake of clarity. 5. Line 286: missing 'c' from co-cultured. 6. Line 311: Check spelling of Ribavirin Staff Comments:

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Reviewer 1
Helgers et al. analyzed the potential role of DCs in EV-A71 dissemination. The experiments are well designed. I have some concerns about the technical issues.
1. Ribavirin could not suppress EV-A71 infection (see Smee et al., 2016, Antiviral Research). Guanidine HCl (2 to 4 mM) or rupintrivir (1 uM) would be useful for the aim. Suppression of EV-A71 infection by these inhibitors must be confirmed to validate the experiments. Therefore, the potential effect of replication in DCs could not be excluded.
As requested, we have now included validation of the inhibitor ribavirin (10 µM) and rupintrivir (1 µM ; 157 -158; 336 -342]. These data support our previous results that viral replication does not occur in DCs.

Protein secretion is inhibited in enterovirus-infected cells (Spagnolo and Kirkegaard, 2009, JVI).
Therefore, a cell surface marker CD1a might not serve as the marker for the EV-A71-infected DCs. To clarify the absence of EV-A71 infection in DCs, the authors should add the data of only DCs, thus in the absence of RD99 cells, in the context of experiments in Fig. 1. figure 1 ; 71 -73; 157 -158; 331 -335]. CD1a is not a secretory protein but a transmembrane protein expressed on the cell surface as well as intracellular by DCs and not downregulated upon activation. In addition, we have determined intracellular and extracellular CD1a expression to ensure detection of CD1a.

Reviewer 2
Helgers et al present a study investigating the role of dendritic cells (DC's) in the transmission of EV-A71. Using a combination of flow cytometry and qRT-PCR the authors demonstrate that, while not susceptible to EV-A71 infection themselves, DC's are capable of transmitting infectious virus to susceptible cells following co-culture, and doing so in a replication-independent manner. They subsequently demonstrate a role for heparan sulfates in this process by inhibiting transmission with soluble heparin. This is an interesting study that suggests a potential mechanism by which EV-A71 is able to establish secondary sites of infection. However, I have some questions/comments to be addressed: Major 1. Did the authors carry out any characterisation of the DC population following differentiation but prior to use in co-culture? DC populations can vary widely between donors and it would be good to include, at minimum, the details of how the DC populations used in co-culture were initially defined. CD1a,CD80,CD83 and CD86 [Suplementary figure 1;[71][72][73][157][158][331][332][333][334][335].

As requested, we have now included a flow cytometry analysis of DCs including the expression of
Minor 1. Line 111: Could the authors clarify the RT reagent used as SupersScript II is not a Promega product.
We have corrected this mistake. The SuperScript II used is from ThermoFischer Scientific.

Line 134: Remove extra full stop after RD99 cells.
We have corrected this mistake. We have corrected this mistake and clarified that CD1a expression is used to distinguish the DCpopulation.
4. Line 185-187: The authors discuss assessing a role for PSGL-1 in DC-mediated transmission of EV-A71. However, there is no data or details in the results on PSGL-1, which instead focus on DC-SIGN and heparan sulfates. Do the authors have this data to include in the manuscript? If not reference to this should be removed from the discussion for the sake of clarity.
We have corrected this mistake and removed this section in the discussion.

Line 286: missing 'c' from co-cultured.
We have corrected this mistake.

Line 311: Check spelling of Ribavirin
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